Gene expression of Nectin‐4 and its clinical significance in dogs with primary lung adenocarcinoma

Abstract Background Canine primary lung adenocarcinoma (CPLA) is suspected by radiography or computed tomography; however, since there are no tumour markers, early diagnosis is difficult, and the prognosis is poor due to increased tumour volume. Nectin‐4 has been reported to be expressed in human lung, ovarian, and pancreatic cancers and promotes tumour growth. It has been reported to be a tumour marker and prognostic factor, and oncolytic virotherapy is being investigated using nectin‐4 as a therapeutic target. Objectives The purpose of this study was to investigate the expression of Nectin‐4 in CPLA and its clinical significance in dogs with pulmonary adenocarcinomas. Methods The relationships between Nectin‐4 expression and signalling, tumour volume, tumour weight, and prognosis were analyzed in 34 CPLA patients. Results The expression of canine Nectin‐4 (high Nectin‐4) was found in 25 of 34 cases (73%), and Nectin‐4 expression levels did not show any significant associations with gender, body weight, and tumour stage. However, there was a significant positive correlation between Nectin‐4 expression and tumour volume (r = 0.623, p < 0.05) and tumour weight (r = 0.735, p < 0.05). Regarding prognosis, the median survival time was 427 days in high Nectin‐4 cases and 420 days in cases with no Nectin‐4 expression. Conclusion Our study demonstrated that Nectin‐4 is highly expressed in CPLA. In addition, nectin‐4 might be a tumour growth factor in CPLA and thus is a promising biomarker for CPLA. Further investigations on nectin‐4 in CPLA are warranted for its diagnosis and novel targets for oncolytic virotherapy.


INTRODUCTION
Canine primary lung tumours are mostly malignant, with adenocarcinoma being the predominant type (Rebhun & Culp, 2013). Clinical symptoms include cough and dyspnoea, but approximately 30% of cases are reported to be asymptomatic (McNeil et al., 1997), and many cases have advanced tumours at the time of diagnosis. In addition, there are no tumour markers, which makes it difficult to detect the tumour at an early stage. Surgical resection is the first choice of treatment, but the prognosis is poor even with surgical resection if metastasis to lymph nodes, other lobes of the lung, or the pleura has occurred (Polton et al., 2008). Furthermore, there are reports that an increase in tumour volume is a factor that worsens the prognosis of canine primary lung adenocarcinoma (CPLA) (Rebhun & Culp, 2013).
These findings suggest that the early diagnosis of CPLA may lead to an improved prognosis.
Nectins and nectin-like molecules (Necls) are calcium-independent immunoglobulin (Ig)-like cell adhesion molecules. Nectins comprise four members (1, 2, 3, and 4), and these have been found to be essential contributors to the formation of cell-cell adhesions (Takai et al., 2008). In addition, they regulate cellular activities such as motility, proliferation, and survival (Takai et al., 2003). Each nectin has a variety of functions and interacts independently or with each other. Nectins-1, 2, and 3 are expressed in various tissues, and nectin-4 has been found to be highly expressed in tissues such as the placenta, vagina, and skin but not in normal lung tissue (Fabre et al., 2002;Reymond et al., 2001). On the other hand, high nectin-4 expression has been found in more than 50% of human breast, pancreatic, and lung cancer patients (Liu et al., 2021). In human pancreatic cancer, the expression of nectin-4 has also been reported to be associated with prognosis (Nishiwada et al., 2015). As a tumour marker, elevated serum nectin-4 has been reported to be useful in human lung cancer (Takano et al., 2009).
In veterinary medicine, thyroid transcription factor-1 (Bettini et al., 2009), which differentiates primary lung carcinomas from metastatic tumours, is being investigated as a tumour marker for canine pulmonary tumours, but it has not been applied in clinical practice.
In veterinary medicine, it is known that canine distemper virus uses nectin-4 as a receptor (Shin et al., 2022), and furthermore, it has been reported that nectin-4 expression is involved in tumour growth in prostate cancer (Salda et al., 2019). However, to our knowledge, no studies have examined the expression of Nectin-4 in CPLA.
We hypothesized that Nectin-4 gene expression in CPLA occurs at a high frequency, similar to that in human lung adenocarcinomas.
In addition, this may lead to the study of tumour markers targeting nectin-4 and the development of treatments with oncolytic virotherapy. The purpose of this study was to determine the gene expression of Nectin-4 and its clinical significance in dogs with pulmonary adenocarcinomas.

Case collection
Canine patients with a pulmonary mass which underwent lung lobectomy and histologically diagnosed as lung adenocarcinoma were included in the study. All patients were investigated for signalment, and blood tests, radiography, and computed tomography (CT) were performed to identify the region of the lung tumour. Staging was based on the classification of canine lung adenocarcinoma (Lee et al., 2020). Consent for the study was obtained from the owners of all the patients.

Surgery
General anaesthesia was pre-administered by subcutaneous injec- Prior to surgery, CT was performed, and the volume (cm 3 ) of the lung mass was measured in terms of length, width, and height. All patients were approached through an intercostal or median sternotomy depending on the region and size of the individual tumours, and the tumours were resected by lung lobectomy. All resected masses were weighed.

Histopathological examination
For histopathological examination, the resected lung masses were sliced and fixed in 10% neutral-buffered formalin and subsequently embedded in paraffin for histopathological diagnosis. Sections (4 µm thick) were deparaffinized in xylene and rehydrated in ethanol to water. The slides were then stained with haematoxylin and eosin and histopathologically evaluated.

Prognosis
The median survival time (MST) was defined as the time from staging to tumour-related death or other causes. The follow-up period was defined as the interval from staging to the last day of followup or death. Additional follow-up information was obtained through telephonic interviews and e-mails with the referring veterinarians.
Canine patients that were still alive at the end of the study were censored.

Control sampling
Normal lung tissue for real-time PCR was collected from five dogs (Beagle, individual numbers: 5FW455, 5FW471, 5FW494, 5FW500, and 5FW504) approved by the ethics committee at our institution.
These dogs underwent blood tests, and radiography revealed that they were healthy. General anaesthesia was pre-administered by subcuta-

RNA isolation
RNA was isolated from the tumours of canine patients and the lung tissues of five healthy dogs (Beagle, control group) using RNA NucleoSpin columns (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Tissue samples were homogenized using a motorized tissue grinder (Thermo Fisher Scientific, Waltham, MA, USA).
The quality and quantity of RNA were determined spectroscopically using a NanoDrop 1000 spectrophotometer (Invitrogen, Carlsbad, CA, USA).

Reverse transcription and quantitative real-time PCR
A high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) was used for cDNA synthesis according to the manufacturer's instructions.

Expression analysis of canine Nectin-4
The expression of canine

Statistical analysis
Correlations between canine Nectin-4 expression and clinical parameters were analyzed using the Mann-Whitney U test and Spearman correlation, and Kaplan-Meier survival curves were plotted and compared using the log-rank test. All statistical analyses were performed using the GraphPad Prism 8.0 software package (GraphPad software, Inc., San Diego, USA).

Case characteristics
Thirty-four dogs were included in this study. Gender was evenly distributed in this cohort of animals: 15 males (12 castrated) and 19 females (18 spayed

Expression and correlation of Nectin-4 in CPLA
High Nectin-4 levels were found in 25 of 34 cases (73%) (Figure 1). A comparison of signalment and other parameters in high Nectin-4 and low Nectin-4 cases is summarized in Table 1. In the cases with the highest expression, there was a marked 107-fold increase compared to the control group. There were no significant differences in canine Nectin-4 expression levels according to body weight, gender, and stage classification ( Figure 2). However, the median tumour volume was 113 cm 3 (3.6-573.8 cm 3 ), and the median tumour weight was 53.1 g (1-285 g).
There was a significant positive correlation between these parameters and canine Nectin-4 expression, with r = 0.623 and r = 0.735, p < 0.05, respectively ( Figure 3).

Prognosis
The MST in high Nectin-4 cases (25 cases) was 427 days and 420 days in low Nectin-4 cases (nine cases), with no significant difference between the groups (Figure 4). Regarding the prognosis by stage, the MST for high Nectin-4 in stage 1 was 718 days and that for low Nectin-4 was TA B L E 1 Comparison of signalment data of high Nectin-4 and low Nectin-4 groups

DISCUSSION
In this study, canine pulmonary carcinomas are likely to express high levels of nectin-4, as reported in human patients. Furthermore, the expression frequency of Nectin-4 in CPLA was as high as 73%. There was no significant difference between canine Nectin-4 expression according to gender, body weight, stage classification, or prognosis, but there was a significant positive correlation between canine Nectin-4 expression level and tumour volume (cm 3 ) and tumour weight (g).
Regarding nectin-4 and the promotion of tumour growth, a mechanism that activates cell motility has been reported. It has been shown that lamellipodia elongation is essential for cell motility and invasion, which requires the activation of the GTPase Rac1. Rac1 is activated by nectin-4 (Takai et al., 2001), and in human lung adenocarcinoma, the expression of nectin-4 activates Rac1 and promotes tumour cell invasion and growth (Takano et al., 2009). It is necessary to analyze whether the expression of Nectin-4 in canine CPLA is involved in tumour growth, as in human Nectin-4-expressing tumours.
The genes for epidermal growth factor receptor and anaplastic lymphoma kinase, which have been found to be mutated in human cancers, have also been studied in canine lung adenocarcinoma (Mariotti et al., 2014), and more recently, human epidermal growth factor receptor 2 protein overexpression in immunohistochemistry has also been found in canine lung cancer (Yoshimoto et al., 2020). Thus, although the characteristics of the cancer are becoming clearer, the diagnosis of CPLA can only be done through radiography or CT scan, with tumour markers not currently clinically applied. On the other hand, cytokeratin 19-fragment (CYFRA21-1) and carcinoembryonic antigen have been used as tumour markers in human lung adenocarcinoma (Dong et al., 2016;Zhao et al., 2014), and reports indicate that sensitivity increases when these markers are measured simultaneously with serum nectin-4 (Takano et al., 2009). In our study, 13 cases (38%) were asymptomatic, and three of the five cases in stage 4 were asymptomatic. Based on these results, it is necessary to increase the use of diagnostic tools such as tumour markers for early diagnosis, as asymptomatic CPLA patients may have stage progression and a worse prognosis. In our study, the high frequency of canine Nectin-4 expression suggested the possibility that nectin-4 may be a useful tumour marker. To our knowledge, there is no ELISA kit that can measure serum nectin-4 levels in dogs; therefore, further research and development are needed.
Nectin-4 has also been found to be a receptor for measles virus (MV) infection (Mühlebach et al., 2011), and MV has been reported to infect tumours expressing nectin-4, inducing tumour regression (Grote et al., 2001 has lost pathogenicity and has been found to have oncolytic effects in human lung adenocarcinoma cells expressing nectin-4 (Fujiyuki et al., 2015). Lung lobectomy is the treatment of choice in CPLA, but the prognosis is still poor even after surgical intervention when there are metastases to other lung lobes or pleura (McNeil et al., 1997). Furthermore, chemotherapy and molecular-targeted drugs had little effect.
Therefore, it is important to study novel therapies for metastatic cases.
Our study may also serve as the basis for the novel treatment of CPLA with rMV-SLAMblind, as Nectin-4 is highly expressed in spontaneous CPLA.
In this study, there was no significant difference between prognosis and canine Nectin-4 expression, even when the cutoff for Nectin-4 expression was more than twofold. Furthermore, the MST in this study by stage classification showed no difference compared to previous reports (Lee et al., 2020). In addition, there were cases with high Nectin-4 expression and large tumour volumes that survived for a long time after surgical resection. Human lung adenocarcinoma is different from the results of our study because nectin-4 expression has been reported to be a poor prognostic factor (Liu et al., 2021;Takano et al., 2009).
However, there is veterinary literature indicating that an increase in tumour volume is a factor that worsens the prognosis of CPLA (Rebhun & Culp, 2013), so further analysis of nectin-4 expression levels and prognosis is necessary.
A limitation of this study was that the expression of canine Nectin-4 was examined genetically, but immunohistochemistry was not performed. Furthermore, the association between histological subtypes of lung adenocarcinoma and Nectin-4 expression levels is important but has not been investigated and needs further investigation. The case with the largest tumour volume and weight was significantly different from the other cases, which had a small effect on the correlation factor. Furthermore, since the number of cases is not large, it is necessary to accumulate more cases and investigate the correlation between disease parameters and canine Nectin-4 expression in the future.
In conclusion, our study demonstrated that Nectin-4 is highly expressed in CPLA and thus is a promising biomarker for CPLA. Further investigations on nectin-4 in CPLA are warranted for its diagnostic utility and as a potential novel target for oncolytic virotherapy.

ACKNOWLEDGEMENT
We would like to thank Editage (www.editage.com) for the English language editing.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
All prodecures and experimental protocols were approved by our Institutional Ethical Committee.

AUTHOR CONTRIBUTIONS
Conceptualization, data curation, investigation, methodology, visualization, writing-original draft, and writing-review and editing: Kei Tamura.